Joshua Daniels (jbdaniel at facstaff.wisc.edu) wrote:
: Hi-
: I've been trying to do plate lysates for DNA extraction for some embl 3 clones
: that I must start mapping!! I've been stuck at this part of the process for a
: month now and I'm going nutso.
: I've been using:
: Top agarose with tryptone, NaCl (lambda medium, per current protocols)
: Lambda bottom agar.
: If I get nice totally lysed lawns after o/n incubation I put 13 ml SM (for a
: 150 mm plate) for 5 hrs and use that as the lystate.
: For getting the DNA I've tryed the following:
: Alternatively, instead of the PEG ppt I simply pelleted phage in an
: ultracentrifuge at 132,000 X g for 1.5 hrs. I got a nice translucent pellet
: which I resuspended in .05 M pH 7.5 Tris.
It's possible that your DNA is still bound to the white goop. Why don't you
try at this point ( prior to phenol chloroform stage) to incubate the
white stuff with TE at 37C for 10-15 minutes. This might entice the DNA to
leave the goop.
Then I added phenol to crack the
: phage. Upon doing this, I got a white ppt which I assume was phage capsid
: protein. The aq phase was phenol ext, and CHCl3 ext several more times and
: then I tried to ppt with 3M NaOAC and 2.5 volumes EtOH (the usual). No dice.
: I got a pellet, but sure wasn't phage DNA.
: Josh
It would be a good idea to also do a dot blot after each stage to keep
track of your DNA. This might tell you which stage is the culprit for the
loss of DNA ( if in fact there is one).
Cheers
--
Shahram Mori _/\_
Program in Molecular Biology _\ /_
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
88003